Quantitative differential proteomic analysis of E. coli O157:H7 clade 8 and clade 6 strains from cattle compared to EDL933 strain

AMIGO NATALIA; QI ZHANG; QUNJIE ZHANG; ZHONGJIAN CHEN; JINLONG BEI; CATALDI ANGEL

Boston

Congreso; VTEC; 2015

Last generation quantitative proteomics allow the identification and the assessment of the differential contents of thousands of proteins in bacterial samples. Our purpose was to identify differentially expressed proteins in two strains of E. coli O157:H7 from cattle and belonging to clade 8 and clade 6 compared to standard EDL933 strain. These isolates from cattle belonging to clade 8 and 6 showed previously to have enhanced pathogenicity in a mice model and virulence-related properties. Cell extracts and culture supernatants of strains Rafaela II (clade 8), 7.1 Anguil (clade 6) and EDL933 were prepared. The proteins were extracted, modified by iodoacetamide, desalted and analyzed using a two dimensional LC and a Thermo Orbitrap Fusion mass spectrometer.In the cell extract 2328 proteins have been found, while 1696 proteins were found in culture supernatants. Except 81 proteins most of the proteins in culture supernatant were also found in cell extract, probably because of cell lysis during culture or centrifugation. The samples were individually labeled by TMT6plex method and database searches were carried out by Mascot to identify the proteins. Scaffold Q+ software was used to determine differentially expressed genes (DGE). Attention was focused on proteins putatively related to virulence. Proteins hyperexpressed in the cell extract were the curli protein CsgC, a transcriptional activator, a putative exported protein, and a serine protein kinase for Rafaela II and a probable lysozyme Inhibitor, a phage protein, the lipoprotein TagA, and Stx2 for 7.1 Anguil. In the case of culture supernatant proteins hyperexpressed were Stx2, three hypothetical proteins, EspC, CheW, and phage proteins for Rafaela II and Stx2, four hypothetical proteins, the lipoprotein TagA for 7.1 Anguil. All these proteins have from 4 to16 fold more abundance in clade 8 and clade 6 than in EDL933 strains.Our next step is to validate these results and to verify the role of these proteins in virulence of E. coli O157:H7.